Test piece and quantitative method and apparatus for an organism-oriented substance

ABSTRACT

A plurality of cDNAs each having a known different base sequence are labeled with a fluorescent dye (FITC), whereby F-cDNA is prepared. The F-cDNA is disposed at a plurality of predetermined positions on the slide glass of a DNA micro array chip. Cy5-cDNA is prepared by synthesizing cDNA from poly(A)-mRNA in the presence of a fluorescent dye (Cy5). The Cy5-cDNA is placed on the DNA micro array chip and hybridized to the F-cDNA. After hybridization, the DNA micro array chip is read by a quantitative apparatus incorporated with an analyzer in which information about the base composition ratio and base length of cDNA has been registered.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to a test piece that is employed indeoxyribonucleic acid (DNA) analysis and immunological analysis and to amethod and apparatus for measuring the quantity of anorganism-originated substance using the test piece.

[0003] 2. Description of the Related Art

[0004] It is now considered possible that the human genome project fordetermining and analyzing all the base sequences of a human genome ashuge as about 3000 Mbp will be completed sooner than the originalscheduled date and determined by 2003, and the focus of the human genomeproject is now shifting from systematic base sequence determination tosystematic function analysis.

[0005] The specific content of generic information comes down to whatprotein is synthesized and under what condition. With respect to theformer, i.e., what protein is synthesized, methods of analysis, such asWestern blotting, Northern blotting, and Southern blotting, are hithertowell known. These methods can analyze what a specific protein, DNA, andribonucleic acid (RNA) sampled are, but are not necessarily suitable foranalyzing all proteins, DNA, and RNA, extracted from a cell, at the sametime.

[0006] On the other hand, regarding the latter, i.e., under whatconditions protein is synthesized, the conventional methods of analysiscannot perform sufficient analysis, because protein is controlled at atransfer level. The main reason for this is that control data of boththe base sequence in DNA and the corresponding contents areinsufficient.

[0007] However, with the latest advancements in techniques for fixingarbitrary oligonucleotide with high density on the surface of a1-centimeter-square carrier called a DNA chip or a DNA micro array chip,it is expected that the analysis of gene expression information willincreasingly advance. The DNA chip is formed by dividing a silicon chipinto a plurality of sections using photolithography and directlysynthesizing single-stranded DNA having a specific base sequence on eachsection. As to the DNA micro array chip, a DNA macro array chip having aspot size of about 300μ or more previously blotted on the membrane isreduced to a spot size of about 200μ or less and blotted on a slideglass. The DNA chip or the DNA micro array chip is connected to a signalreader and a computer system, and it can be known which probe DNA ishybridized by the DNA disposed on the chip or the micro array chip.Depending on the DNA type disposed on the DNA chip or the DNA microarray chip and the disposition, it is possible to employ the chip or thearray chip in various analyses such as DNA mutation analysis, DNApolymorphism analysis, DNA base sequence analysis, and DNA expressionanalysis.

[0008] The analysis employing the DNA micro array chip, however, stillhas quite a number of problems because discussions about array chipgeneration and a detector thereof have only just started. For instance,the micro array chip is made by blotting complementary DNA (cDNA) bymeans of a spotter, and as a method of generating the micro array chip,there is a contact printing method and a non-contact printing method. Inthe contact printing method, cDNA 43 is disposed on a slide glass 42 bya pin 41 in direct contact with the slide glass 42, as shown in FIG. 4A.In the non-contact printing method, cDNA 43 is blotted on the slideglass 42 by a syringe 44 in non-contact with the slide glass 42, asshown in FIG. 4B. In both the printing methods, however, there is adifference in quantity between blotted spots. Even in the best case,there is a quantity difference of 5 to 10%CV for the contact printingmethod and a quantity difference of 3 to 5%CV for the non-contactprinting method. Further, there are sometimes defect spots and spoiledspots. For this reason, DNA varies in quantity between spots a and b ona DNA micro array chip 151, as shown in FIG. 5. DNA also varies inquantity between spot a on the DNA micro array chip 51 and spot a on aDNA micro array chip 152 generated in the same way. Because of thisdifference in quantity, there is a problem that the quantitativeanalysis of different DNAs generated from a single cell and thequantitative comparison of DNAs in the same cell differing in quantityof expression at a different time will actually include a considerableerror.

[0009] To solve this problem, an improvement in the spotter wasinitially considered, but improvements to enhance the reproducibility ofthe quantity of a sample to be spotted are considered to have limits.

SUMMARY OF THE INVENTION

[0010] The present invention has been made in view of the aforementionedcircumstances. Accordingly, it is the object of the present invention toprovide a test piece, such as a DNA micro array chip, which is capableof performing accurate quantitative analysis even when there is adifference in quantity between blotted spots, without relying on animprovement in a spotter which enhances the reproducibility of theblotted spots.

[0011] To achieve the aforementioned object of the present invention andin accordance with one aspect of the present invention, there isprovided a test piece for analyzing an organism-originated substancelabeled with a second labeling substance. The test piece comprises acarrier on which a plurality of known specific binding substancesdiffering from one another are disposed at a plurality of predeterminedpositions. The specific binding substances are labeled with a firstlabeling substance.

[0012] Since a plurality of known specific binding substances differingfrom one another are disposed at a plurality of predetermined positionson a carrier and labeled with a labeling substance, the quantity of aspecific binding substance disposed on the test piece can be specifiedregardless of a difference in quantity between the specific bindingsubstances that is caused when they are disposed on the carrier.

[0013] In accordance with another aspect of the present invention, thereis provided a quantitative method comprising the steps of:

[0014] detecting a level of a first labeling signal emitted by a firstlabeling substance, which labels a plurality of known different specificbinding substances respectively disposed at a plurality of predeterminedpositions on a carrier of a test piece, for each of the plurality ofpredetermined positions;

[0015] binding an organism-originated substance, labeled with a secondlabeling substance differing from the first labeling substance, to thespecific binding substance and detecting a level of a second labelingsignal emitted from the second labeling substance for each of theplurality of predetermined positions; and

[0016] measuring a quantity of the organism-originated substance boundto the specific binding substance, based on the detected level of thefirst labeling signal and the detected level of the second labelingsignal.

[0017] In accordance with still another aspect of the present invention,there is provided a quantitative apparatus comprising:

[0018] first detection means for detecting a level of a first labelingsignal emitted by a first labeling substance, which labels a pluralityof known different specific binding substances respectively disposed ata plurality of predetermined positions on a carrier of a test piece, foreach of the plurality of predetermined positions;

[0019] second detection means for detecting a level of a second labelingsignal emitted by a second labeling substance, which differs from thefirst labeling substance and labels an organism-originated substancebound to the specific binding substance, for each of the plurality ofpredetermined positions; and

[0020] analyzing means for measuring a level of the organism-originatedsubstance bound to the specific binding substance, based on the detectedlevel of the first labeling signal and the detected level of the secondlabeling signal.

[0021] According to the quantitative method and the quantitativeapparatus of the present invention, a level of a first labeling signalemitted by a first labeling substance, which labels a plurality of knowndifferent specific binding substances respectively disposed at aplurality of predetermined positions on a carrier of a test piece, isdetected for each of the plurality of predetermined positions. Anorganism-originated substance, labeled with a second labeling substancediffering from the first labeling substance, is bound to the specificbinding substance and a level of a second labeling signal emitted by thesecond labeling substance is detected for each of the plurality ofpredetermined positions. Also, a quantity of the organism-originatedsubstance bound to the specific binding substance is measured based onthe detected level of the first labeling signal and the detected levelof the second labeling signal. Therefore, it is possible to measure thequantity of the organism-originated substance independently of adifference in quantity between the specific binding substances. Inaddition, it becomes possible to read out the specific binding substanceand the organism-originated substance disposed on the same test piece atthe same time, because the labeling substance for theorganism-originated substance differs from the labeling substance forthe specific binding substance.

[0022] Note that if the test piece, the quantitative method, and thequantitative apparatus of the present invention are employed, aneffective selection of medicine and wide utilization such as functionalanalysis of EST will become possible, for example, by measuring variousproteins manifested according to the growth of a cancer, with theexplication of the control contents and mechanism of protein synthesisbeing controlled at a transfer level or the realization of themeasurement of a specific protein synthesized in the process of adisease, obtained from messenger RNA (mRNA) transferred within a cell.

[0023] The “carrier” may be any type if a specific binding substance canbe stably bound and spotted. For example, the carrier may be a membranefilter, a slide glass plate, etc. These carriers may be preprocessed tostably bind a specific binding substance.

[0024] The “specific binding substance” means a substance bindablespecifically with an organism-originated substance, such as hormones, atumor marker, enzyme, an antibody, an antigen, abzyme, the otherproteins, a nucleic acid, cDNA, DNA, RNA and the like. The “known”varies depending on the specific binding substance. For example, in thecase of a nucleic acid, the “known” means that the base sequence and thebase length are known, and in the case of protein, it means that thecomposition of the amino acid is known. Here, the specific bindingsubstances disposed at predetermined positions on the carrier means thatone kind of specific binding substance has been disposed for eachposition.

[0025] The “organism-originated substance” is a substance thatspecifically binds with a known specific binding substance disposed at apredetermined position on the carrier, and means, for example,substances extracted, isolated and the like from a living organism. The“organism-originated substance” includes substances extracted directlyfrom a living organism and also includes these substances chemicallyprocessed and chemically modified. For instance, the“organism-originated substance” includes hormones, a tumor marker,enzyme, an antibody, an antigen, abzyme, the other proteins, a nucleicacid, cDNA, DNA, RNA and the like.

[0026] The specific binding substance labeled with a labeling substance(also referred to simply as a labeled specific binding substance) may belabeled at one point such as one end of stranded molecules or at a fewpoints. If the specific binding substance has been labeled at one point,the quantity of the specific binding substance disposed at each positionon the carrier is usually detectable. In the case where enhancement ofdetection sensitivity is desired or in the case where it is technicallydifficult or becomes technically complicated to label the specificbinding substance at one point, the specific binding substance may belabeled at a few points.

[0027] It is preferable that the labeling substance for theorganism-originated substance is different from the labeling substancefor the specific binding substance. The reason for this is that alabeling signal from the labeling substance for the organism-originatedsubstance and a labeling signal from the labeling substance for thespecific binding substance can be detected independently of each otherat the same time. The labeling substance for the organism-originatedsubstance may label the organism-originated substance at one point or ata few points; although one point is preferred. The reason for this isthat in the case of the organism-originated substance, there are caseswhere its component is not known and therefore confirmation of a methodof taking a labeling substance into the organism-originated substancebecomes necessary and technically complicated. Note that in the case ofa known labeling substance, the organism-originated substance may belabeled at a few points similarly to the specific binding substance.

[0028] The “labeling substance” means a marker substance that changeseither a portion of the specific binding substance or a portion of theorganism-originated substance, or is added directly to these substances,in order to obtain information from these substance. The labelingsubstance is not particularly limited, as long as a detection signalemitted therefrom can be detected and also a rule that the labelingsubstance is taken into either the specific binding substance or theorganism-originated substance is known in advance. For example, it ispreferable to employ a fluorescent dye such as SYBR Green II™, Cy5™,fluorescein isothiocyanate and the like, or a radioactive isotope suchas ³²P, ³³P and the like. The “labeling signal” means one emitted oroutput from a labeling substance. For example, the labeling signal meansfluorescent light when the labeling substance is a fluorescent dye andradiation when the labeling substance is a radioactive isotope. In thiscase, a radioactive isotope may be employed in a specific bindingsubstance and a fluorescent dye in an organism-originated substance, ora fluorescent dye may be employed in a specific binding substance and aradioactive isotope in an organism-originated substance. Furthermore,fluorescent dyes may be employed in both a specific binding substanceand an organism-originated substance. Note that in the case wherefluorescent dyes are employed in both a specific binding substance andan organism-originated substance, it is necessary to employ fluorescentdyes whose fluorescent wavelength bands do not overlap with each other.When they overlap, it is necessary to employ fluorescent dyes that donot overlap at at least a major band of detection. On the other hand,when a radioactive isotope is employed, a specific binding substance ona carrier labeled with the radioactive isotope is contacted with aphotostimulable phosphor sheet and is exposed and the sheet is read by alaser, as disclosed in Japanese Patent Publication No. 5(1993)-20712(automatic radiography of measuring a quantity of a radioactiveisotope). The “rule that a labeling substance is taken into either aspecific binding substance or an organism-originated substance is knownin advance” means that when the labeling substance is SYBR Green II™,for example, there is a rule that it is weakly bound to single-strandedDNA or RNA and absorbed in accordance with the base length. Also, whenthe labeling substance is Cy5-nucleotide, there is a rule that it istaken randomly or into an end of DNA or RNA. On the other hand, aradioactive isotope such as ³²P varies depending on a substance that islabeled by the radioactive isotope. For instance, when dNTP[α-³²P],which is employed as substrates in synthesizing cDNA from mRNA, is used,there is a rule that ³²P is randomly incorporated and the amount of ³²Pis in proportion to the base included in the labeled nucleotide.

[0029] The “binding an organism-originated substance to the specificbinding substances” means a case (hybridization) such that a stabledouble strand, as is viewed in DNA or RNA, is formed betweencomplementary nucleotides and also means an extremely high specificitybond that selectively reacts only to a specific substance, such as abond between an antigen and an antibody, a bond between biotin andavidin and the like.

[0030] The “measuring a quantity of the organism-originated substancebound to the specific binding substance, based on the detected level ofthe first labeling signal and the detected level of the second labelingsignal” means that because the level of the first labeling signalemitted from the first labeling substance of the specific bindingsubstance at one position on the carrier is proportional to the quantityof the specific binding substance disposed at that position, the levelof the second labeling signal of the second labeling substance of theorganism-originated substance bound to the specific binding substance iscaused to correspond to the level of the first labeling signal, andtherefore the quantity (density) of the organism-originated substancecan be measured regardless of a difference in quantity between thespecific binding substances. For example, consider the case where thespecific binding substance is cDNA. Assume that when the number of cDNAs(specific binding substances) with one end labeled with a fluorescentdye is s at the nth position on the carrier, the quantity of fluorescentlight (quantity of a labeling signal emitted from a labeling substance)is Ps. On the other hand, the number of probe DNAs (organism-orientedsubstances) with one end of one molecule labeled with a fluorescent dyeis assumed to be c. Furthermore, assume that when the probe DNA and thenth cDNA are hybridized, the quantity of fluorescent light is Pc. It hasbeen said that in a liquid phase system, probe DNA which is hybridizedwith cDNA depends on cDNA and conditions, but is about 1/100. Althoughit cannot be said that the case of the carrier is a perfect liquid phasesystem, the density m of the probe DNA is proportional at least to thenumber of cDNAs existing at the nth position, s. Therefore, Pc isproportional to Pc and m and the following relationship is established.

Pc∞mPs

[0031] Therefore, if Ps and Pc are measured, the density m of theorganism-originated substance bound at the nth position will beobtained.

[0032] In the case where cDNA is labeled at one point, the value of Pshas no relation to the base sequence and base length of cDNA and isproportional only to the number of cDNAs disposed at the nth position.However, in the case where a labeling substance labels a specific baseor labels a few points (in certain cases, tens of points) per onemolecule of cDNA, the quantity of fluorescent light is varied even bythe base length, and consequently, even if all the positions on a sheetof carrier indicate the same quantity of fluorescent light, it cannot besaid that cDNA of the same quantity has been blotted at all thepositions, because the bases of cDNA vary in length. Therefore, in thiscase, there is a further need to calculate “a characteristic valuerelated to cDNA”. That is, a characteristic value related to cDNA, suchas the base composition ratio and base length of cDNA disposed on thecarrier, a quantity of fluorescent light when N cDNAs are included atone position on a carrier and the like, needs to be registered in acomputer for each position. What characteristic value becomes necessaryvaries depending on a labeling substance employed. The fluorescencequantity Pc of probe DNA is proportional to the density m of probe cDNAand the fluorescent quantity Ps of cDNA and inversely proportional tothe number of labeling substances labeling single-stranded cDNA.Therefore, the density m of probe DNA is represented by the followingequation, and a characteristic value that can calculate the number oflabeling substances labeling single-stranded (1 molecule) cDNA becomesnecessary.

m∞Pc/Ps×(number of a labeling substance labeling single-stranded cDNA)

[0033] For instance, in the case of labeling one of the 4 bases of cDNA,the number of labeled specific bases included in 1-molecule cDNA becomesnecessary as a characteristic value. In the case where a labelingsubstance is proportional to the length of a base, the base length andthe base rate at which 1 labeling substance is incorporated (i.e., howmany of every of the bases 1 labeling substance is absorbed) becomenecessary as characteristic values.

[0034] The above and many other objects, features and advantages of thepresent invention will become manifest to those skilled in the art uponmaking reference to the following detailed description and accompanyingdrawings in which preferred embodiments incorporating the principle ofthe present invention are shown by way of illustrative example.

BRIEF DESCRIPTION OF THE DRWAINGS

[0035]FIG. 1 is a flowchart showing how a test piece according to anembodiment of the present invention is made,

[0036]FIG. 2 is a schematic diagram showing an embodiment of aquantitative apparatus of the present invention,

[0037]FIG. 3 is a diagram showing data held in the quantitativeapparatus of the present invention,

[0038]FIG. 4A is a side view showing how cDNA is disposed on a slideglass by a pin in direct contact with the slide glass,

[0039]FIG. 4B is a side view showing how cDNA is blotted on the slideglass by a syringe in non-contact with the slide glass, and

[0040]FIG. 5 is a perspective view of conventional DNA micro arraychips.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Referring to FIG. 1, there is shown a preferred embodiment of atest piece in accordance with the present invention. In the preferredembodiment, a DNA micro array chip is used as an example of the testpiece and cDNA is used as an example of a specific binding substance.Furthermore, mRNA extracted from a cell is used as an example of anorganism-originated substance.

[0042] In a DNA micro array chip 10 according to the preferredembodiment, a plurality of different cDNAs 1 (specific bindingsubstances) each having a known base sequence are labeled with afluorescent dye 5 (e.g., fluorescein isothiocyanate (hereinafterreferred to as FITC)) and are disposed at predetermined positions on aslide glass (carrier) 3. The surface of the slide glass 3 ispreprocessed with a poly-N-lysine solution, and the fluorescent dye 5 isa labeling substance which labels a specific binding substance.

[0043] The cDNA 1 is prepared from known DNA, mRNA and the like using aPCR method or a RT-PCR method. At this time, if deoxycytidine5′-triphosphate (dCTP) labeled with the FITC 5 is employed, the positionof cytosine (C) of the 4 bases of DNA is labeled with the FITC 5,whereby F-cDNA (labeled specific binding substance) 2 can be prepared.The prepared F-cDNA 2 is spotted at predetermined positions on the slideglass 3 by a spotter, thus making the DNA micro array chip 10.

[0044] On the other hand, mRNA (organism-originated substance) 4 to bemeasured is extracted from a cell, and poly(A)-mRNA having apolyadenylic acid (poly(A)) tail at its 3′ end is extracted from mRNA 4.If cDNA is synthesized from poly(A)-mRNA in the presence of Cy5-dUTP(e.g., Cy5 which labels an organism-originated substance), Cy5-cDNA(probe DNA) is made. Of course, it is possible to label the end by useof a labeling primer.

[0045] The Cy5-cDNA is prepared in a predetermined solution and isslowly placed on the DNA micro array chip 10 to perform normalhybridization.

[0046] Now, with reference to FIG. 2, a description will be given of aquantitative apparatus 100 that measures the Cy5-cDNA hybridized on theDNA micro array chip 10 shown in FIG. 1. The quantitative apparatus 100includes (1) a sample tray 20 on which the DNA micro array chip 10distributing the F-cDNA 2 labeled with the FITC 5 is placed at apredetermined position; (2) an argon (Ar) laser (excitation wavelength488 nm) or a SHG laser (excitation wavelength 473 nm) 21 which emitslaser light L1 of luminescence wavelength suitable for exciting thefluorescent dye (FITC) 5; (3) a He—Ne laser (excitation wavelength 633nm) or a semiconductor laser (excitation wavelength 635 nm) 22 whichemits laser light L2 of luminescence wavelength suitable for excitingthe fluorescent dye (Cy5) 6; (4) a first dichroic mirror 23 whichtransmits the first laser light L1 therethrough and reflects the secondlaser light L2; (5) a photomultiplier (hereinafter referred to as a PMT)90 which photoelectrically detects fluorescent light emitted from thefluorescent dyes 5, 6 on the DNA micro array chip 10 excited by thefirst laser light L1 and the second laser light L2; (6) an optical head50 which directs the first laser light L1 and the second laser light L2emitted from the first and second lasers 21, 22 to the DNA micro arraychip 10 placed on the sample tray 20 and also guides the fluorescentlight emitted from the DNA micro array chip 10 to the PMT 90; (7) afilter set 80 with two kinds of switchable band-pass filters 81, 82disposed in the optical path between the optical head 50 and the PMT 90;(8) horizontal scanning means 60 which moves the optical head 50 atuniform speed in the direction of arrow X; (9) vertical scanning means70 which moves the lasers 21, 22, the optical head 50, the filter set80, and the PMT 90 as one body in the direction of arrow Y perpendicularto the direction of arrow X; (10) an amplifier 91 which logarithmicallyamplifies a detection signal detected by the PMT 90; (11) an A/Dconverter 92 which converts the amplified detection signal to a digitalsignal; (12) an analyzer 93 which analyzes the digital signal bycomparing the digital signal with the previously input data on the DNAmicro array chip 110; and (13) a control unit 95 which controls emissionof the first laser light L1 and the second laser light L2 and alsocontrols the filter set 80 so that either the band-pass filter 81 or theband-pass filter 82 is disposed in the above-mentioned optical path.

[0047] Next, a description will be given of the operation of thequantitative apparatus 100 of the preferred embodiment.

[0048] The DNA micro array chip 10, which includes the F-cDNA 2 labeledwith the FITC 5 and the Cy5-cDNA hybridized to the F-cDNA 2, is firstplaced on the sample tray 20. The control unit 95 controls the first andsecond lasers 21, 22 so that the first laser light L1 and the secondlaser light L2 are selected and emitted. As a result of this control,the first laser 21 emits the first laser light L1, while the secondlaser 22 emits the second laser light L2. The control unit 95 alsocontrols the filter set 80 so that the first filter 81 is disposed inthe optical path between the optical head 50 and the PMT 90. In thisway, the filter set 80 places the first filter 81 in the optical path.

[0049] The first laser light L1 emitted from the first laser 21 istransmitted through the dichroic mirror 23 and travels in the directionof arrow X. The first laser light L1 incident on the plane mirror 51 ofthe optical head 50 is reflected upward. The reflected light beam L1passes through the aperture 52 a of an aperture mirror 52 and isincident on a lens 53. A small area on the DNA micro array chip 10placed on the sample tray 20 is irradiated with the first laser lightL1. On the other hand, the optical head 50 is being moved at high anduniform speed in the direction of arrow X by the horizontal scanningmeans 60, so that the first laser light L1 scans the DNA micro arraychip 10 in the direction of arrow X. During this horizontal scanning,for the F-cDNA 2 on the small area irradiated with the first laser L1,the FITC 5 is excited by the first laser light L1 and emits fluorescentlight K1.

[0050] The fluorescent light K1 emitted by the first laser light L1spreads in all directions from the lower surface of the DNA micro arraychip 10 and is formed into a downward fluorescent light beam K1 by thelens 53 of the optical head 50. The fluorescent light beam K1 isreflected by the reflecting surface of the aperture mirror 52 andtravels in the direction of arrow X. The first band-pass filter 81prevents the passage of light other than the fluorescent light beam K1,so only the fluorescent light beam K1 traveling in the direction ofarrow X is incident on the PMT 90. The fluorescent light beam K1incident on the PMT 90 is amplified and photoelectrically detected as acorresponding electrical signal by the PMT 90. The electrical signal isamplified by the logarithmic amplifier 91 and is converted to a digitalsignal by the A/D converter 92. The digital signal is output to theanalyzer 93.

[0051] If single horizontal scanning ends in this manner, the opticalhead 50 is returned to the original position by the horizontal scanningmeans 60. While the optical head 50 is being returned to the originalposition, the vertical scanning means 70 moves the lasers 21, 22, theoptical head 50, the filter set 80, and the PMT 90 as one body in thedirection of arrow Y. By reiterating the horizontal scanning and thevertical scanning, the entire surface of the DNA micro array chip 10 isirradiated with the first laser light L1, and the fluorescent light beamK1 corresponding to each position on the DNA micro array chip 10 isacquired as a digital signal.

[0052] If the data of the fluorescent light beam K1 is acquired up tothe last position on the DNA micro array chip 10 by the horizontalscanning and the vertical scanning, the optical head 50 is returned tothe initial position. Similarly, a fluorescent light beam K2 is emittedfrom the DNA micro array chip 10 irradiated with the second laser lightL2 emitted from the second laser 22 and is incident on the PTM 90 by thesecond band-pass filter 82 which prevents the passage of light otherthan the fluorescent light beam K2. Next, the fluorescent light beam K2is digitized and acquired by repeating the horizontal scanning and thevertical scanning in the same way as the above-mentioned fluorescentlight beam K1. In this embodiment, although the fluorescent light beamK1 is first read out for all the positions on the DNA micro array chip10 and then the fluorescent light beam K2 is read out, the operation offirst emitting the first laser light L1 to read out the fluorescentlight K1 and then emitting the second laser light L2 to the sameposition to read out the fluorescent light K2 may be repeated for allthe positions on the DNA micro chip array 10.

[0053] As shown in FIG. 3, the data of the base composition ratio(adenine (A), guanine (G), cytosine (C), thymine (T)) persingle-stranded F-cDNA disposed at each position on the DNA micro arraychip 10 has been registered in the analyzer 93 that has acquired thedigital signal corresponding to each position on the DNA micro arraychip 10. Therefore, the density of Cy5-cDNA at each position can becalculated from the registered data of the base composition ratio persingle-stranded F-cDNA, the registered data of the fluorescent lightquantity of F-cDNA, the measured fluorescent light quantity of F-cDNA,and the measured fluorescent light quantity of Cy5-cDNA. For instance,if the fluorescent light quantity of F-cDNA at the (1-1)st position isassumed to be P₁ and the fluorescent light quantity of Cy5-cDNA at the(1-1)st position is assumed to be P₂, the density m of Cy5-cDNA willbecome m∞P₂/P₁×20. If analysis is likewise performed for all thepositions on the DNA micro array chip 10, the density of Cy5-cDNA ateach position will be obtained.

[0054] In the preferred embodiment, although FITC and Cy 5 have beenemployed as fluorescent dyes, it is also possible to employ the otherfluorescent dyes and radio isotopes. In this case, if the known baselength and base composition ratio of cDNA and the known fluorescentlight or radiation quantity of cDNA at each position have beenregistered according to a labeling substance in the analyzer 93,calculating the density of probe DNA at each position will be possibleby measuring either the fluorescent light or radiation quantity of cDNAon a newly made DNA micro array chip and measuring either thefluorescent light or radiation quantity of probe DNA.

[0055] While the present invention has been described with the DNA microarray chip as a test piece, cDNA as a specific binding substance, andmRNA extracted from a cell as an organism-originated substance, theinvention is not limited to this example, but may be modified within thescope of the appended claims.

What is claimed is:
 1. A test piece for analyzing an organism-originatedsubstance labeled with a first labeling substance, the test piececomprising: a carrier on which a plurality of known specific bindingsubstances differing from one another are disposed at a plurality ofpredetermined positions; wherein said specific binding substances arelabeled with a second labeling substance.
 2. The test piece as set forthin claim 1 , wherein said first labeling substance and said secondlabeling substance differ from each other.
 3. The test piece as setforth in claim 1 , wherein said specific binding substances arecomplementary deoxyribonucleic acid (cDNA).
 4. The test piece as setforth in claim 2 , wherein said specific binding substances are cDNA. 5.The test piece as set forth in claim 1 , wherein said second labelingsubstance is a fluorescent dye.
 6. The test piece as set forth in claim2 , wherein said second labeling substance is a fluorescent dye.
 7. Thetest piece as set forth in claim 3 , wherein said first labelingsubstance is a fluorescent dye.
 8. The test piece as set forth in claim4 , wherein said second labeling substance is a fluorescent dye.
 9. Thetest piece as set forth in claim 1 , wherein said second labelingsubstance is a radioactive isotope.
 10. The test piece as set forth inclaim 2 , wherein said second labeling substance is a radioactiveisotope.
 11. The test piece as set forth in claim 3 , wherein saidsecond labeling substance is a radioactive isotope.
 12. The test pieceas set forth in claim 4 , wherein said second labeling substance is aradioactive isotope.
 13. A quantitative method comprising the steps of:detecting a level of a first labeling signal emitted by a first labelingsubstance, which labels a plurality of known different specific bindingsubstances respectively disposed at a plurality of predeterminedpositions on a carrier of a test piece, for each of said plurality ofpredetermined positions; binding an organism-originated substance,labeled with a second labeling substance differing from said firstlabeling substance, to said specific binding substances and detecting alevel of a second labeling signal emitted from said second labelingsubstance for each of said plurality of predetermined positions; andmeasuring a quantity of said organism-originated substance bound to saidspecific binding substance, based on the detected level of said firstlabeling signal and the detected level of said second labeling signal.14. The quantitative method as set forth in claim 13 , wherein saidspecific binding substances are cDNA.
 15. The quantitative method as setforth in claim 13, wherein said measurement is further made based on acharacteristic value related to cDNA.
 16. The quantitative method as setforth in claim 14 , wherein said measurement is further made based on acharacteristic value related to cDNA.
 17. The quantitative method as setforth in claim 13 , wherein said first labeling substance for saidspecific binding substances is a fluorescent dye.
 18. The quantitativemethod as set forth in claim 14 , wherein said first labeling substancefor said specific binding substances is a fluorescent dye.
 19. Thequantitative method as set forth in claim 15 , wherein said firstlabeling substance for said specific binding substances is a fluorescentdye.
 20. The quantitative method as set forth in claim 16 , wherein saidfirst labeling substance for said specific binding substances is afluorescent dye.
 21. The quantitative method as set forth in claim 13 ,wherein said first labeling substance for said specific bindingsubstances is a radioactive isotope.
 22. The quantitative method as setforth in claim 14 , wherein said first labeling substance for saidspecific binding substances is a radioactive isotope.
 23. Thequantitative method as set forth in claim 15 , wherein said firstlabeling substance for said specific binding substances is a radioactiveisotope.
 24. The quantitative method as set forth in claim 16 , whereinsaid first labeling substance for said specific binding substances is aradioactive isotope.
 25. A quantitative apparatus comprising: firstdetection means for detecting a level of a first labeling signal emittedby a first labeling substance, which labels a plurality of knowndifferent specific binding substances respectively disposed at aplurality of predetermined positions on a carrier of a test piece, foreach of said plurality of predetermined positions; second detectionmeans for detecting a level of a second labeling signal emitted by asecond labeling substance, which differs from said first labelingsubstance and labels an organism-originated substance bound to saidspecific binding substance, for each of said plurality of predeterminedpositions; and analyzing means for measuring a quantity of saidorganism-originated substance bound to said specific binding substance,based on the detected level of said first labeling signal and thedetected level of said second labeling signal.
 26. The quantitativeapparatus as set forth in claim 25 , wherein said specific bindingsubstances are cDNA.
 27. The quantitative apparatus as set forth inclaim 25 , wherein said analyzing means further performs saidmeasurement, based on a characteristic value related to cDNA.
 28. Thequantitative apparatus as set forth in claim 26, wherein said analyzingmeans further performs said measurement, based on a characteristic valuerelated to cDNA.
 29. The quantitative apparatus as set forth in claim 25, wherein said first labeling substance for said specific bindingsubstances is a fluorescent dye.
 30. The quantitative apparatus as setforth in claim 26 , wherein said first labeling substance for saidspecific binding substances is a fluorescent dye.
 31. The quantitativeapparatus as set forth in claim 27 , wherein said first labelingsubstance for said specific binding substances is a fluorescent dye. 32.The quantitative apparatus asset forth in claim 28 , wherein said firstlabeling substance for said specific binding substances is a fluorescentdye.
 33. The quantitative apparatus as set forth in claim 25 , whereinsaid first labeling substance for said specific binding substances is aradioactive isotope.
 34. The quantitative apparatus as set forth inclaim 26 , wherein said first labeling substance for said specificbinding substances is a radioactive isotope.
 35. The quantitativeapparatus as set forth in claim 27 , wherein said first labelingsubstance for said specific binding substances is a radioactive isotope.36. The quantitative apparatus as set forth in claim 28 , wherein saidfirst labeling substance for said specific binding substances is aradioactive isotope.